The overall goal of our research is to identify the molecular cues that influence motor axons as they navigate long distances to their target muscles in the limb and define how they work mechanistically. The results of our studies should yield important insights into the factors that control neural regeneration and repair following disease or trauma. This proposal is a logical extension of our previous studies and focuses on the function of EphA4/ephrins, c- ephexin and c-Ret. Our general hypothesis is that these factors act at multiple steps in the development of motor axon projections to the limb, as directional cues involved in pathway selection, general promoters of growth or as stimulators of axon fasciculation. The specific aims of our studies are as follows: 1. Test the hypothesis that distinct ephrins in axons are vital for axon fasciculation, growth, and dorsoventral pathway selection. 2. Test the hypothesis that ephrins and EphA4 regulate segmental growth, limb entry, and pathway selection. 3. Test the hypothesis that c-ephexin is required in motor axons to select their muscle targets correctly. 4. Test the hypothesis that c-Ret acts as a positive factor to promote motor axon pathway selection of a dorsal trajectory. In chicken embryos, effects on axon fasciculation, growth, pathway selection and muscle target selection wiill be analyzed following loss- and gain-of-function manipulations targeted precisely to motor neurons, somitic mesoderm, and limb mesoderm using in ovo electroporation. Our strong preliminary studies demonstrate clearly that shRNA against c-ephexin reduces protein levels in vitro and in vivo, and results in phenotypic defects in the timing of axon entry into the limb. These data strongly support the usefulness of this approach in the proposed studies.